Agrobacterium-mediated transformation of sunflower (Helianthus annuusL.): a simple protocol

Abstract

A simple and reproducibleAgrobacterium tumefaciens-mediated transformation system was developed for sunflower (Helianthus annuusL.) 'KBSH-1'. The objective was to substantially eliminate thein vitroregeneration component from the transformation protocol. Two-d-old seedlings with one cotyledon detached were infected withAgrobacterium tumefaciensstrain LBA 4404/pKIWI105 harbouring genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II). Following co-cultivation, seedlings were grown aseptically for 5 d on a growth regulator-free basal medium supplemented with 250 μ g ml⁻¹cefotaxime (a bacteriostat used to control gram negative bacteria). Seedlings were screened for transient GUS expression and the shoot portions of the putative transformants were utilized for propagation as transgenic plants. The excised shoots that initiated roots following selection were subsequently transferred to a glasshouse. Molecular analysis of transgenic plants confirmed concordance between the presence of foreign genes and enzyme activity. The transformation regime facilitated rapid generation of up to 2% phenotypically normal fertile plants containing functional transgenes. The transmission and integration of the marker genes in the progeny is demonstrated.